The DNA synthesis is employed for various purposes in the research of the field of genetic engineering. Almost all of them, except for synthesis of short chain DNA such as oligonucleotides, are carried out by the enzymatic method utilizing DNA polymerase. Accordingly, the DNA polymerase is highly valuable for reagents for DNA sequencing, DNA labelling, or site-directed mutagenesis. In addition, recently, thermostable DNA polymerases have been remarked with the developments of the polymerase chain reaction (PCR) method and the reverse transcription-PCR(RT-PCR) in which the PCR method and the reverse transcriptase reaction are combined. Therefore, various kinds of DNA polymerases suitable for PCR method mentioned above have been developed, and commercialized.
The presently known DNA polymerases can be roughly classified by amino acid sequence homology into four families, among which Family A (pol I type enzymes) and Family B (α-type enzymes) account for the great majority. Although the DNA polymerases belonging to the respective families possess generally similar biochemical properties, detailed comparison reveals that depending upon individual enzymes, each of the DNA polymerases has different properties for substrate specificity; substrate analog-incorporating efficiency; degree and rate for primer extension; mode of DNA synthesis; presence or absence of exonuclease activity; optimum reaction conditions such as temperature and pH, and sensitivity against inhibitors. Therefore, the enzyme best suited for the application has so far been selected from the available DNA polymerases.
For example, Pyrococcus furiosus, a hyperthermophilic archaebacterium, produces DNA polymerase belonging to α-type, and the gene thereof has been isolated [Nucleic Acids Research 21, 259-265 (1993)]. Recently, novel DNA polymerase showing no structural similarity to any known DNA polymerase was found in the above bacterial strain. In this DNA polymerase, two novel proteins form a complex, whereby exhibiting DNA polymerase activity. In addition, the novel DNA polymerase exhibits potent 3′→5′ exonuclease activity and excellent primer extension activity. For example, when the enzyme is used for PCR, a DNA fragment of a size of about 20 kb can be amplified.
On the other hand, in DNA synthesis reaction using DNA polymerase, it is important to set appropriate reaction conditions, as well as to select an appropriate enzyme. Major conditions for reaction include reaction mixture composition, pH, reaction temperature, and template and primer concentration. In addition, these reaction conditions must be set in accordance with the enzyme used and the purpose. However, such settings may be difficult to make in some cases.
In addition, there has been known that efficient DNA synthesis can be carried out by using a combination of plural DNA polymerases, wherein the efficient DNA synthesis could not be achieved by single DNA polymerase [Proc. Natl. Acad. Sci. USA 91, 5695-5699 (1994)]. The method is a method using in PCR a mixture of DNA polymerase having 3′→5′ exonuclease activity (for example, the above Pyrococcus furiosus-derived α-type DNA polymerase) and DNA polymerase not having such an activity (for example, Thermus aquaticus-derived DNA polymerase (Taq DNA polymerase), and is known as LA-PCR method. According to this method, there are exhibited such effects that the yield of amplified DNA is increased, as compared with that of conventional PCR using only one kind of DNA polymerase, and that long chain length of DNA which could not be amplified by conventional PCR can be amplified. However, such effects are only exhibited when an enzyme having 3′→5′ exonuclease activity is used in combination with an enzyme having no such activity.
As described above, the DNA synthesis reaction using DNA polymerase is indispensable as a genetic engineering procedure. Moreover, it is important to increase their efficiency for research or the like. However, a presently available reaction system has a defect in that it is not sufficiently optimized to be utilized for research or the like. For this reason, there has been a demand for a method enabling more efficient DNA synthesis as compared to that of the conventional DNA synthesis reactions.